SHV-28, an extended spectrum β-lactamase from a clinical isolate of Klebsiella pneumoniae , had an isoelectric point of 7.6 and a substrate profile showing preferential hydrolysis for cefotaxime over ceftazidime. It differed from SHV-1 by one amino acid substitution. The conserved S-T-F-K and K-T-G motifs were identified by SHV-28 protein sequencing.